Interaction between a Sulfated Polysaccharide from Sea Cucumber and Gut Microbiota Influences the Fat Metabolism in Rats

Due to its significant physiological effects, a sulfated polysaccharide has been considered an important nutrient of sea cucumber, but its metabolism in vivo is still unclear. The present study investigated the metabolism of a sea cucumber sulfated polysaccharide (SCSP) in rats and its influence on the metabolite profiles. The quantification by HPLC-MS/MS revealed that the blood level of SCSP achieved a maximum of 54.0 ± 4.8 μg/mL at 2 h after gavage, almost no SCSP was excreted through urine, and 55.4 ± 29.8% of SCSP was eliminated through feces within 24 h. These results prove the utilization of SCSP by gut microbiota, and a further microbiota sequencing analysis indicated that the SCSP utilization in the gut was positively correlated with Muribaculaceae and Clostridia_UCG-014. In addition, the non-targeted metabolomic analysis demonstrated the significant effects of SCSP administration on the metabolite profiles of blood, urine, and feces. It is worth noting that the SCSP supplement decreased palmitic acid, stearic acid, and oleic acid in blood and urine while increasing stearic acid, linoleic acid, and γ-linolenic acid in feces, suggesting the inhibition of fat absorption and the enhancement of fat excretion by SCSP, respectively. The present study shed light on the metabolism in vivo and the influence on the fat metabolism of SCSP.


Introduction
Sea cucumber is valuable seafood, and it has been considered as a functional food and consumed traditionally in Asian countries, especially in China, Japan, and Korea [1].A sea cucumber sulfated polysaccharide (SCSP) is the critical nutrient in sea cucumber, and it consists of two sulphated polysaccharides, namely fucosylated chondroitin sulphate and fucoidan sulphate [1,2].The fucosylated chondroitin sulphate has a chondroitin core and a unique sulfated fucose side chain [3].Fucoidan sulphate from sea cucumber is a linear polysaccharide consisting of α1→3 linked fucose repeating units with various sulfation patterns [3].SCSP has significant physiological effects such as anticancer [4], antibacterial activity [5], hypolipidemic [6], immune regulation [3,7] and anti-inflammation [8].However, the underlying mechanism of its effects in vivo is still unclear.It has been proved that SCSP cannot be digested by gastrointestinal enzymes or acid liquid [9,10].Moreover, as a macromolecule, it is difficult for SCSP to travel through intestinal epithelial cells, which could be inferred through the studies on other macromolecules [11].Thus, more efforts are needed to reveal the action pathway of SCSP after oral supplementation.
Recently, more and more reports have verified that oral administration of indigestible polysaccharides could regulate gut microbiota so as to benefit the host health [12].Our previous report has revealed that SCSP can effectively prevent diet-induced obesity through modulating the composition of gut microbiota [13].It has been proposed that indigestible polysaccharides can be metabolized by gut microbiota to produce bioactive metabolites, such as short-chain fatty acids, demonstrating beneficial effects on host health [14].However, little evidence for the polysaccharide consumption by gut microbiota in vivo has been reported.Sulfated polysaccharides are more resistant to gut microbiota due to their sulfate groups [15,16].Our previous study found that a part of SCSP could be fermented by fecal microbiota in vitro.But the utilization degree of SCSP by gut microbiota in vivo is still unknown.
Metabolomics have been widely used to analyze what occurs in diseases' process by identifying potential biomarkers and the related metabolic pathway [17].However, increasing evidence has suggested that some metabolites produced by gut microbiota have potent functions for host energy metabolism [18,19].Non-targeted metabolomics are a comprehensive approach for detecting metabolites as much as possible, and it is more robust in characterizing the metabolism profile and finding involved metabolites without targets [20].Thus, in the present study, nontargeted metabolomics have been applied to monitor the regulation of SCSP on the metabolites in blood, urine, and feces to reveal the underlying action mechanism of SCSP.
The present study aimed to demonstrate the absorption, metabolism, and excretion of sea cucumber polysaccharides in vivo, and to analyze the effects of SCSP administration on the metabolite profiles of blood, urine, and feces, thus revealing the action pathway of sea cucumber polysaccharides in vivo.

Animals
All animal experiments were performed in accordance with the guidelines for care and use of laboratory animals of Dalian Polytechnic University, and the experiment was approved by the Animal Ethics Committee of Dalian Polytechnic University (ID DLPU2018008).
Eighteen male Sprague Dawley (SD) rats (300 ± 20 g) were purchased from Liaoning Changsheng Biotechnology Company (Benxi, China).The rats were kept under standard conditions with a 12 h light-dark cycle, 23 ± 2 • C ambient temperature, and 55 ± 10% relative humidity.The feed was composed of corn, soybean meal, flour, bran, fish flour, salt, calcium hydrogen phosphate, rock flour, multivitamins, multiple trace elements, amino acids, etc., and more details are shown in Supplemental Table S5.The rats were acclimatized in clean cages for a fortnight prior to the experiment and weighed (310 ± 20 g), and then randomly divided into two groups: the Control group (n = 9) and the SCSP group (n = 9, named S1~S9).
To prevent the effects of food on SCSP absorption, the rats were fasted for 12 h before SCSP administration but drank water freely.The rats in the SCSP group were gavaged with 150 mg/kg of the SCSP solution (2 mL), and the Control group were gavaged with the same volume of water.Then, the rats were put into metabolic cages and fed normally after 4 h.Fecal and urine samples were collected at 24 h and 48 h (frozen at −80 • C) to ensure that SCSP was wholly excreted.After 1 day, 150 mg/kg of the SCSP solution was gavaged again.Then, the blood was extracted from the tail of the rats at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h after administration, and placed in centrifuge tubes containing an EDTA anticoagulant.At 24 h, the rats were anesthetized with ether, and blood samples were collected from the orbital vein and placed in centrifuge tubes containing the EDTA anticoagulant and ordinary centrifuge tubes.The coagulation time of the blood collected at 2 h after gavage from the broken tail of rats using a capillary was measured according to previously described reports [21,22].

Quantification of SCSP in Plasma, Feces, and Urine
Firstly, SCSP in plasma and feces was isolated.Plasma samples (0.1 mL) were suspended in a mixture of 0.5 mL of 2.6 M trifluoroacetic acid (TFA) and 0.4 mL of water.Dried fecal samples (0.20 g) were dissolved in 4 mL of water, and after centrifugation at 8000 rpm for 10 min, the supernatant (1 mL) was collected and mixed with ethanol (4 mL) and kept at 4 • C overnight.Then, the precipitate was collected after centrifugation (4000 rpm, 10 min).
Quantification of SCSP was conducted as previously described [2].Briefly, the collected precipitate containing SCSP was redissolved in water (5 mL).After that, 0.5 mL of the solution was mixed with 0.5 mL of 2.6 M TFA.Urine samples (10 mL) were concentrated to 1 mL using a rotary evaporator at 50 • C under a vacuum.Then, a four-fold volume of ethanol was added, and the mixture stood at 4 • C overnight.After that, the precipitate was collected by centrifugation (4000 rpm, 10 min), and dissolved in 1 mL of 1.3 M TFA.
Immediately after the above treatment, the samples were heated at 105 • C for 3 h in sealed tubes for the acid hydrolysis.Then, the resulting solutions were dried with a vacuum concentrator (LaboGene Aps., Lynge, Denmark).Then, 0.5 mL of methanol was added to the solution, which was dried again.This procedure was repeated thrice to remove TFA thoroughly.Then, 100 µL of 1 mg/mL lactose was added as an internal standard, and the mixtures were resolved in a solution composed of 400 µL of ammonia and 400 µL of a 0.3 M PMP methanolic solution.After heating at 70 • C for 30 min in a water bath, the solution was dried with the vacuum concentrator.Thus, the PMP derivatives of hydrolysates of SCSP were prepared for an ESI-MS/MS analysis.
The chromatography separation was carried out on a Shimadzu HPLC system (LC-20ADXR pumps, SIL-20AXP Autosampler, Kyoto, Japan) with a Thermo Scientific (Waltham, MA, USA) Hypersil Gold column (150 × 2.1 mm, 5 µm).A 20 mM ammonium acetate solution and acetonitrile (85:15, v/v) were used as the mobile phase at a flow rate of 0.5 mL/min, and the column temperature was maintained at 30 • C. The ESI-MS/MS analysis was performed on a Q-trap 4000 triple-quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA) in the positive-ion multiple reaction monitoring (MRM) mode.Mass spectrometry analysis parameters are provided in Supplemental Table S4.

Sequencing Analysis of the Gut Microbiota
Sequencing was carried out on an Illumina MiSeq PE250 platform (Novogene Genomics Technology Co., Ltd., Beijing, China, https://magic.novogene.com(accessed on 1 June 2021)).Briefly, Genomic DNA was isolated from fecal samples by the sodium dodecyl sulfate (SDS) method and assayed using agarose gel electrophoresis.After that, the genomic DNA was amplified with the 341F (5 -CCTACGGGNGGCWGCAG-3 ) and 806R (5 -GGACTACHVGGGTWTCTAAT-3 ) primers specific for the V3-V4 region of the 16S rRNA gene.Sequencing libraries were generated using the Ion Plus Fragment Library Kit 48 rxns and assessed on the Qubit@ 2.0 fluorometer (Thermo Scientific, Waltham, MA, USA).Alpha diversity and rarefaction curves were analyzed using QIIME V1.7.0.Venn diagrams were constructed using R software package V3.0.3 and were used to compare and analyze differences in the composition of OTUs in the intestinal flora between groups.A principal coordinate analysis of PCoA and β-diversity was calculated using QIIME software V1.7.0 and presented using Stat, Ggplot2, and WGCNA packages in R software V2.15.3.
UPLC-Q-TOF MS analyses were performed on AB Sciex Triple TOF 5600 using both positive ion (PI) and negative ion (NI) modes.The chromatography separation was carried out using Waters Xselect @HSS T3 (2.5 µm, 100 mm × 2.1 mm) at a flow rate of 0.4 mL/min, and the column temperature was maintained at 30 • C. For the PI mode, the mobile phase was eluent A (0.1% formic acid in water, v/v) and eluent B (acetonitrile with 0.1% formic acid, v/v).For the NI mode, the mobile phase was eluent A (5 mM ammonium acetate in water) and eluent B (5 mM ammonium acetate in water/acetonitrile = 10:90, v/v).The solvent gradient was set as follows: 0-5 min, 0-20% B; 5-7 min, 20% B; 7-14 min, 20-100% B. The quality Control samples (QC) were prepared by mixing equal (10 µL) amounts of each serum sample.The AB Sciex triple TOF 5600 mass spectrometer was operated with ion source gas 1 of 55 arb, ion source gas 2 of 55 arb, curtain gas of 35 arb, a temperature of 550 • C, and ion spray voltage floating of 5500 V for the PI mode and −4500 V for the NI mode.
The collected raw data were converted to "abf" format and the converted files were imported into MSDIAL.A principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed with SIMCA 14.1 software after exportation.And then the database in MSDIAL was used to identify the metabolites with a VIP value greater than 1.0.

Statistical Analysis
All data are expressed as means ± standard deviations (SDs).Student's t-test was used to evaluate the difference between two groups, and three groups were made by one-way ANOVA with Duncan's test.p < 0.05 was considered to be significantly different.A multiple linear regression analysis was used to investigate the relationship between the utilization of SCSP and bacterial OTUs.Bacterial OUTs with >1% abundance were included in the regression model.

Absorption and Exertion of SCSP in Rats
In order to reveal the metabolism of SCSP in rats, SCSP in plasma, feces, and urine of rats after the gavage (46.5 mg) was quantified by HPLC-MS/MS.The method for the quantification of SCSP was validated for sensitivity, recovery, and repeatability (Supplemental Results).The results showed that the blood concentration of SCSP reached the maximum (54.0 ± 4.8 µg/mL) at 2 h (Figure 1A).Since the circulating blood volume of rats is generally about 11 mL, it could be estimated that 1.2% SCSP was in blood circulation at that time.As shown in Table 1, SCSP in urine samples collected within 0~24 h and 24~48 h was nearly undetectable, and no more than 0.2% SCSP was excreted through urine as polymers.The same phenomenon has been previously reported [24].Of note, 55.4 ± 29.8% of SCSP was detected in fecal samples within 24 h although there was great variability among individuals (Figure 1B).However, SCSP was barely detectable in feces after 24 h.From the above results, it could be concluded that the SCSP was mainly eliminated via feces within Foods 2023, 12, 4476 5 of 15 24 h.Interestingly, a considerable proportion (~44%) of SCSP disappeared, indicating its degradation by intestinal flora.It has been reported that SCSP could be partially degraded by intestinal flora [25][26][27].Moreover, the differences of SCSP contents in feces among rat individuals could be contributed to their diversity of intestinal microbiota communities [28].As shown in Figure 1C, SCSP significantly prolonged coagulation time compared to the Control group (p < 0.01).Nadezhda E. Ustyuzhanina and R.J.C. Fonseca et al. had proved that fucosylated chondroitin sulfate in sea cucumber had an obvious anticoagulant effect by in vitro and in vivo experiments [29,30].Our experiments also demonstrated the anticoagulant effect of gavaged SCSP, indicating that SCSP could be absorbed into the systemic circulation.
detected in fecal samples within 24 h although there was great variability among uals (Figure 1B).However, SCSP was barely detectable in feces after 24 h.From th results, it could be concluded that the SCSP was mainly eliminated via feces with Interestingly, a considerable proportion (~44%) of SCSP disappeared, indicating radation by intestinal flora.It has been reported that SCSP could be partially degr intestinal flora [25 -27].Moreover, the differences of SCSP contents in feces amon dividuals could be contributed to their diversity of intestinal microbiota communi As shown in Figure 1C, SCSP significantly prolonged coagulation time compare Control group (p < 0.01).Nadezhda E. Ustyuzhanina and R.J.C. Fonseca et al. had that fucosylated chondroitin sulfate in sea cucumber had an obvious anticoagula by in vitro and in vivo experiments [29,30].Our experiments also demonstrated coagulant effect of gavaged SCSP, indicating that SCSP could be absorbed into temic circulation.

Relationship between Intestinal Flora and SCSP Utilization
The gut microbiota of the rats were analyzed through multiplex sequencing covering the V3-V4 regions of 16S rRNA, and the differences in the microbiota of the rat individuals were demonstrated.The relative abundances of microbiota at the phylum level (Figure 2A) showed that Bacteroidetes and Firmicutes were the dominant intestinal bacteria in these rats.The composition of Bacteroidetes was further demonstrated as shown in Figure 2B.Muribaculaceae was the most abundant family in Bacteroidetes, with an abundance of 85.2%, followed by Prevotellaceae (~10%) and Bacteroideae (~1%).Furthermore, the relative contributions of 35 dominant genera showed in the heatmap (Figure 2C) revealed that the composition of the intestinal flora varied greatly among individuals.The correlation between Operational Taxonomic Units (OTUs) and the utilization rate of SCSP was demonstrated by a multiple linear regression analysis (Supplemental Table S3) to obtain the regression model y = 35.164x 1 + 38.257x 2 − 0.145, where x 1 and x 2 are from Muribaculaceae and Clostridia_UCG-014, respectively.Thus, the result indicates the two OTUs were both positively correlated with the utilization rate of SCSP (p < 0.05).SCSP metabolism is a complex process that requires the participation of many gut microbes.It has been reported that some microorganisms such as Bacteroides and Parabacteroides are also involved in the metabolism of SCSP [27].Different gut microbiota structures could respond to the intake food differently [27,31].The discrepancy in the SCSP utilization in these rats could be attributed to the great diversity of their microbiota communities.Bacteroidetes, including Muribaculaceae, possess very large numbers of genes encoding carbohydrate-active enzymes, and are considered as the main contributors for polysaccharide utilization [32,33].Muribaculaceae widely exist in the gut microbiota and a genome analysis indicated their potential capability to degrade complex carbohydrates [34].Clostridia_UCG-014 has been identified as a member of Clostridiaceae, which possessed some well-studied cellulolytic organisms [35], and it has been considered as one of the major microbial contributors to digest polysaccharides due to its carbohydrate-degrading genes [36].Then, the present study will suggest some bacteria from Muribaculaceae and Clostridiaceae that are keystone bacteria for SCSP utilization in the gut, affecting SCSP's outcome, such as anti-obesity and anti-type 2 diabetes [37].

Regulation of SCSP on Metabolite Profile of Feces
The effect of SCSP administration on the fecal metabolites was investigated through a non-targeted metabolomic analysis using Q-TOF-MS.As shown by PCA (Figure 3A) and OPLS-DA (Supplemental Figure S3), the fecal metabolite profiles of the SCSP group and the Control group separated with each other, indicating SCSP changed the metabolic profiles in the host gut.In total, 95 differential metabolites were identified in feces (Tables 2 and 3), and the related metabolic pathways with -log10 (p) and impact as the horizontal and vertical coordinates are shown in Figure 3B.The metabolic pathway obviously influenced by the SCSP supplement includes biosynthesis of unsaturated fatty acids, pantothenate and CoA biosynthesis, β-alanine metabolism, aminoacyl-tRNA biosynthesis, primary bile acid biosynthesis, riboflavin metabolism, linoleic acid metabolism, purine metabolism, histidine metabolism, retinol metabolism, arginine and proline metabolism, pyrimidine metabolism, tyrosine metabolism, and steroid hormone biosynthesis.Interestingly, the biosynthesis of unsaturated fatty acids and linoleic acid metabolism were both related to lipid metabolism.Of note, stearic acid, linoleic acid, and γ-linolenic acid in these two pathways were enriched by SCSP, indicating the enhancement of lipid metabolism in the guts of rats administrated SCSP. the biosynthesis of unsaturated fatty acids and linoleic acid metabolism were both related to lipid metabolism.Of note, stearic acid, linoleic acid, and γ-linolenic acid in these two pathways were enriched by SCSP, indicating the enhancement of lipid metabolism in the guts of rats administrated SCSP.
As we know, diet fat is digested and absorbed in the stomach and the small intestine [38].More active lipid metabolism in the guts suggests more fat was transported to the gut without being absorbed by the upper digestive tract.Thus, the results in the present study indicate the supplementation of SCSP could inhibit the fat absorption.It has been reported that SCSP could prevent obesity induced by a high-fat diet [13], and the inhibition effect of SCSP against the fat absorption is one of the possible reasons for its antiobesity activity.Liu et al. reported that the gut microbiota in HFD-fed mice were dominated by Muribaculaceae species, and the untargeted metabolomics analyses of fecal samples show that microbial changes altered the bacteria-derived metabolites, which related to linoleic acid metabolism [39].Thus, it can be inferred that SCSP could interfere with linoleic acid metabolism by affecting Muribaculaceae to exert anti-obesity effects.As we know, diet fat is digested and absorbed in the stomach and the small intestine [38].More active lipid metabolism in the guts suggests more fat was transported to the gut without being absorbed by the upper digestive tract.Thus, the results in the present study indicate the supplementation of SCSP could inhibit the fat absorption.It has been reported that SCSP could prevent obesity induced by a high-fat diet [13], and the inhibition effect of SCSP against the fat absorption is one of the possible reasons for its anti-obesity activity.Liu et al. reported that the gut microbiota in HFD-fed mice were dominated by Muribaculaceae species, and the untargeted metabolomics analyses of fecal samples show that microbial changes altered the bacteria-derived metabolites, which related to linoleic acid metabolism [39].Thus, it can be inferred that SCSP could interfere with linoleic acid metabolism by affecting Muribaculaceae to exert anti-obesity effects.

Regulation of SCSP on Metabolite Profiles of Serum and Urine
To evaluate the physiological effect of single gavage of SCSP, the metabolites in serum and urine of the rats were also determined by UPLC-Q-TOF MS in both positive and negative modes.As shown in Tables 4-7, 17 and 39 differential metabolites were identified in the serum and urine, respectively.The PCA analysis demonstrated a distinction between the serum metabolite profiles of the Control and SCSP groups in both of the positive (Figure 4A) and negative modes (Figure 4B).Moreover, although no significant separation was observed in urine profiles of the Control and SCSP groups detected in the positive mode (Figure 4C), the analysis result determined in the negative mode that an obvious separation was exhibited (Figure 4D).The separation between Control and SCSP groups demonstrated in the negative mode could be attributed to some acidic compounds, such as glycolic acid and arachidic acid.The metabolism pathway analysis in serum and urine metabolites is shown in Supplemental Figure S4.Thus, it can be concluded that single-dose SCSP administration can affect the in vivo metabolism of rats.Of note, palmitic acid, stearic acid, and oleic acid, which were all increased in the feces of the SCSP group, all decreased in serum and urine of the SCSP group.These long-chain fatty acids (LCFAs) are components of pork fat in the fodder, and they could be absorbed into serum [40].Thus, the less serum LCFAs in the SCSP group indicated that SCSP could inhibit the LCFAs' absorption so as to increase fat excretion via feces.It has been reported that the dietary supplementation of SCSP could significantly reduce fat accumulation and lipid levels in high-fat-diet-fed mice [41].Then, the findings in the present study suggest the inhibition of LCFAs' absorption plays a role in the anti-obesity and hypolipidemic effects of SCSP.It has been well documented that dietary fibers could prevent the absorption of fat by chelation with their micro-molecular chains [42].Then, it could be inferred these SCSP micromolecules may bind to fat in the intestine so as to interfere with the fat metabolism in the host.
Taking together all the above results, single gavage of SCSP in vivo could be proposed as follows: a proportion of SCSP could participate in various metabolic reactions of the rats to produce different metabolites, thus benefiting the host health.The other SCSP was not degraded, but they could reduce obesity and hyperlipidemia by inhibiting fat absorption and increasing fat excretion into the feces.This is consistent with the previous research result that SCSP can prevent diet-induced obesity and its associated diseases [13].

Conclusions
The present study demonstrated the absorption, the exertion, and the metabolism by gut microbiota of SCSP.As shown by the quantitative analysis of SCSP in blood, urine, and feces, only a small amount of SCSP could be absorbed into the blood while a proportion of SCSP was excreted in the feces, which suggests that a considerable proportion of SCSP was metabolized by gut microbiota and the metabolized amount of SCSP varied among individuals.The SCSP degradation in the intestine was positively correlated with Muribaculaceae and Clostridia_UCG-014, confirming the involvement of these gut bacteria in metabolism of SCSP in the gut.Furthermore, the changes of the metabolites in blood, urine, and feces caused by SCSP administration revealed that SCSP could inhibit the fat absorption by increasing fat excretion.Our findings provide a better understanding of the action mechanism of SCSP in vivo.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/foods12244476/s1.Supplemental Results 1 The validation of the method for quantification of SCSP.Supplemental Table S1 The LOD and LOQ of solid samples (dried sea cucumber) and liquid samples (sea cucumber wine).Supplemental Table S2 The recoveries of SCSP in solid samples (dried sea cucumber), liquid samples (sea cucumber wine), defatted feces, and undefatted feces.Supplemental Table S3 The coefficient of the regression model, which is about the correlation between OTUs with abundances ≥ 1% and the utilization rate of SCSP.Supplemental Table S4 Mass spectrometry analysis parameters.Supplemental Table S5 Feed components (per kg of feed).Supplemental Figure S1 MRM chromatogram of PMP-fucose in plasma samples from the Control group (A) and the SCSP group (B), in urine samples from the Control group (C) and the SCSP group (D), and in fecal samples from the Control group (E) and the SCSP group (F).Supplemental Figure S2 The standard curve of PMP-fucose.Supplemental Figure S3 OPLS-DA of metabolites in feces detected in the positive (A) and negative (B) modes.Supplemental Figure S4 The metabolism pathway analysis in serum (A) and urine (B) metabolites.

Figure 1 .
Figure 1.Mean plasma concentration-time profile of SCSP in rats after the gavage (n = 3, excreted proportions of SCSP through feces within 24 h in different rat individuals (B), and of SCSP on coagulation time in rats (n = 3, ** p < 0.01, (C)).S1 to S9 stand for 9 rats m different individuals.Graph bars marked with different letters on top represent statisticall cant results (p < 0.05) based on one-way analysis of variance (ANOVA) followed by Dunc

Figure 1 .
Figure 1.Mean plasma concentration-time profile of SCSP in rats after the gavage (n = 3, (A)), the excreted proportions of SCSP through feces within 24 h in different rat individuals (B), and the effect of SCSP on coagulation time in rats (n = 3, ** p < 0.01, (C)).S1 to S9 stand for 9 rats making up different individuals.Graph bars marked with different letters on top represent statistically significant results (p < 0.05) based on one-way analysis of variance (ANOVA) followed by Duncan's test, whereas bars labelled with the same letter correspond to results that show no statistically significant differences.

Foods 2023 , 16 Figure 2 .
Figure 2. The relative abundances of intestinal microflora in different rats at the phylum level (A), the relative abundances of different families in Bacteroidota (B), and the heatmap of top 35 genera (C).

Figure 2 .
Figure 2. The relative abundances of intestinal microflora in different rats at the phylum level (A), the relative abundances of different families in Bacteroidota (B), and the heatmap of top 35 genera (C).

Figure 3 .Table 2 .Figure 3 .
Figure 3. PCA analysis of the feces metabolites detected in the positive (A) and negative (B) modes and metabolism pathway analysis in rat feces (C).

Figure 4 .
Figure 4. PCA analysis of the serum and urine metabolites detected in the positive (A,C) and negative (B,D) modes.

Figure 4 .
Figure 4. PCA analysis of the serum and urine metabolites detected in the positive (A,C) and negative (B,D) modes.

Author
Contributions: Y.Z.: Investigation, Data curation, Writing-original draft.H.S.: Formal analysis.Z.L.: Data curation.C.A.: Methodology.C.Y.: Writing-review and editing.X.D.: Project administration.S.S.: Conceptualization, Writing-original draft, Supervision, Project administration, Funding acquisition.All authors have read and agreed to the published version of the manuscript.

Table 1 .
Excreted amounts and ratios of SCSP in feces and urine.
a The data were not significantly different from those in Control group.

Table 2 .
Identification results of differential metabolites in feces detected in the positive ion mode.

Table 3 .
Identification results of differential metabolites in feces detected in the negative ion mode.

Table 4 .
Identification results of differential metabolites in serum detected in the positive ion mode.Differential metabolites were analyzed using unpaired two-tailed Student's t-test with Bonferroni correction (* p < 0.05, and *** p < 0.001 vs. the Control group; ↑, increase; ↓, decrease).

Table 5 .
Identification results of differential metabolites in serum detected in the negative ion mode.

Table 6 .
Identification results of differential metabolites in urine detected in the positive ion mode.

Table 7 .
Identification results of differential metabolites in urine detected in the negative ion mode.

Table 4 .
Identification results of differential metabolites in serum detected in the positive ion mode.